Quinolones are widely used antimicrobial agents because of their broad antibacterial spectrum and reliable action against otherwise resistant bacteria. Quinolone resistance has been slowly but steadily increasing. It has been previously shown that various chromosomal mutations contribute to this resistance, but plasmid-mediated resistance has not been shown to occur. The Principal Investigator has discovered a 56 kb, broad host range, multiresistance plasmid in clinical isolates of Klebsiella pneumoniae and Escherichia coli that confers quinolone resistance in a variety of gram negative bacteria. The level of plasmid-mediated quinolone resistance is low in wild-type E. coli, but higher degrees of quinolone resistance can be selected for when the plasmid is present. The quinolone resistance (qnr) locus has been cloned and sequenced from the 56 kb plasmid. The nucleotide sequence is unique, but the predicted protein (termed Qnr) has similarity to the microcin B17 immunity protein which protects DNA gyrase from microcin inhibition. The aims of the proposal are to identify the qnr promoter by sequencing the 5' end of its mRNA, to construct a high level expression and purification system for Qnr, to study the ability of purified Qnr to protect DNA gyrase and topoisomerase IV from quinolone inhibition, to explore binding of Qnr to these quinolone targets and their subunits, and to establish the prevalence of qnr mediated resistance, both at the hospital where it was first detected and in samples of resistant bacteria from other sources. Understanding how qnr acts may help to curtail the rise in quinolone resistance and may also provide fundamental insights into aspects of DNA replication.